DNA methylation assays for body fluid identification

ABSTRACT

The invention pertains to analyzing the DNA methylation levels at specific genetic loci. The DNA methylation levels at the specific genetic loci can be used to detect the presence in a sample of a specific body fluid, for example, semen containing sperm, saliva, or blood. Particularly, the DNA methylation levels at the genetic loci corresponding to SEQ ID NOs: 1, 7, 13, and 19 are used to detect sperms, saliva or blood cells. The DNA methylation levels at the specific loci can be determined by high-resolution melt (HRM) analysis or sequencing of the amplicons produced using specific primers designed to amplify the specific loci. Kits containing the primers and reagents for carrying out the methods disclosed herein are also provided.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a divisional application of U.S. Ser. No.16/712,411, filed Dec. 12, 2019, which is incorporated herein byreference in its entirety.

SEQUENCE LISTING

The Sequence Listing for this application is labeled “SeqList-ST25,”which was created on Dec. 12, 2019, and is 8 KB. The Sequence Listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Human body fluids such as blood, saliva, semen, or sweat are commonlyencountered at crime scenes. Determination of the type and origin ofbody fluids can provide valuable insights into the circumstances leadingto the deposition of the DNA evidence at the crime scene. Body fluidsare also very useful in crime scene reconstruction. Currently, bodyfluid identification relies on serological or immunological methods thatare based on colorimetric detection of protein markers. Because of theunspecific presence of many of these markers in different body fluids,several of the current methods are presumptive and cannot confirm thepresence of the suspected body fluids. In addition, some of the methodsmay require a large sample and even destroy the sample during testing,both of which are impractical for forensic specimens.

BRIEF SUMMARY OF THE INVENTION

In contrast to the conventional methods, the instant invention providesDNA methylation markers that provide highly tissue specific andsensitive analysis for identification of source body fluids,particularly, semen containing sperms, saliva, and blood. Thesemethylation markers can also be used to determine the origins of the DNAsamples, either from a single or multiple sources. In certainembodiments, the methods disclosed herein can be used for theidentification of semen containing sperms, saliva, and blood usingpyrosequencing and/or high-resolution melt (HRM) analysis. Thus, theinvention provides methods that avoid the problems and difficultiesassociated with the current methods of detecting body fluids,particularly, forensic samples.

In one embodiment, the body fluid and/or cells present in a samplecomprise semen or sperm, saliva or buccal epithelial cells, and/or bloodor blood cells.

Assays for determining the DNA methylation levels at specific loci inthe genomic DNA isolated from a sample are provided. In certainembodiments, the assays comprise HRM analysis or pyrosequencing ofamplicons produced using specific primers designed to amplify specificgenetic loci in the genomic DNA. Kits containing primers and reagentsfor performing the methods disclosed herein are also provided.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 provides a chart showing the mean percent of methylation on theNMUR2 locus determined by pyrosequencing for samples of blood (n=23),saliva (n=24), sperm (n=20), and vaginal secretion (n=22), ±standarddeviation of the mean.

FIG. 2 provides a chart showing the mean percent of methylation on theUBE2U locus determined by pyrosequencing for samples of blood (n=23),saliva (n=24), sperm (n=20), and vaginal secretion (n=22), ±standarddeviation of the mean.

FIG. 3 provides a chart showing the mean percent of methylation usingthe SA-6 locus determined by pyrosequencing for samples of blood (n=23),saliva (n=24), sperm (n=20), and vaginal secretion (n=21), +/−standarddeviation of the mean.

FIG. 4 provides a chart showing the mean percent of methylation on theAHRR locus determined by pyrosequencing for samples of blood (n=23),saliva (n=24), sperm (n=20), and vaginal secretion (n=22), ±standarddeviation of the mean.

FIG. 5 provides melt curves from samples amplified and analyzed for theNMUR2 locus showing that the melting temperatures for semen containingsperms is lower than those of other body fluids (semen with no sperms,vaginal secretion, blood, and saliva) (n=3 for each sample).

FIG. 6 provides a graph showing the mean values for melting temperatures(° C.) for the NMUR2 locus obtained by HRM analysis for samples of sperm(Tm=80.9° C.), vaginal secretion (84.6° C.), saliva (84.5° C.), andblood (84.5° C.), ±standard deviation.

FIG. 7 provides melt curves from samples amplified and analyzed with theUBE2U locus showing that the melting temperatures for semen containingsperms is lower than those of other body fluids (semen with no sperms,vaginal secretion, blood, and saliva) (n=3 for each sample).

FIG. 8 provides a graph of the mean values for melting temperatures (°C.) for the UBE2U marker obtained by HRM analysis for samples of sperm(n=22, Tm=77.1° C.), vaginal secretion (n=20, Tm=78.7° C.), saliva(n=21, Tm=78.8° C.) and blood (n=20, Tm=78.7° C.), ±standard deviation.

FIG. 9 shows melt curves from samples amplified and analyzed with SA-6marker showing melting temperatures for saliva samples are higher thanthe melting temperatures for the samples of the other body fluids (semenwith no sperms, semen containing sperms, vaginal secretion, and blood)(n=3 for each sample).

FIG. 10 shows graph for the mean values for melting temperatures (° C.)for SA-6 marker obtained by HRM analysis for saliva samples (n=22,Tm=77.6° C.), vaginal secretion (n=21, Tm=76.5° C.), sperm (n=19,Tm=76.4° C.) and blood (n=21, Tm=76.4° C.), +/−standard deviation.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1: Sequence of the NMUR2 locus specific for sperm.

SEQ ID NO: 2: Sequence of a forward primer designed to amplify thebisulfite treated NMUR2 locus.

SEQ ID NO: 3: Sequence of a reverse primer designed to amplify thebisulfite treated NMUR2 locus.

SEQ ID NO: 4: Sequence of a sequencing primer designed to sequence theamplicon produced from the bisulfite treated NMUR2 locus.

SEQ ID NO: 5: Sequence of the amplicon produced from the NMUR2 locusafter bisulfite treatment assuming 0% methylation of all CpG sites. Allcytosines, including cytosines from CpG sites are converted to thymines.

SEQ ID NO: 6: Sequence of the amplicon produced from the NMUR2 locusafter bisulfite treatment assuming 100% methylation of all CpG sites.Cytosines other than the cytosines from the CpG sites are converted tothymines. Cytosines from the CpG sites remain as cytosines.

SEQ ID NO: 7: Sequence of the UBE2U locus specific for sperm.

SEQ ID NO: 8: Sequence of a forward primer designed to amplify thebisulfite treated UBE2U locus.

SEQ ID NO: 9: Sequence of a reverse primer designed to amplify thebisulfite treated UBE2U locus.

SEQ ID NO: 10: Sequence of a sequencing primer designed to sequence theamplicon produced from the bisulfite treated UBE2U locus.

SEQ ID NO: 11: Sequence of the amplicon produced from the UBE2U locusafter bisulfite treatment assuming 0% methylation of all CpG sites. Allcytosines, including cytosines from CpG sites are converted to thymines.

SEQ ID NO: 12: Sequence of the amplicon produced from the UBE2U locusafter bisulfite treatment assuming 100% methylation of all CpG sites.Cytosines other than the cytosines from the CpG sites are converted tothymines. Cytosines from the CpG sites remain as cytosines.

SEQ ID NO: 13: Sequence of the AHRR locus specific for blood.

SEQ ID NO: 14: Sequence of a forward primer designed to amplify thebisulfite treated AHRR locus.

SEQ ID NO: 15: Sequence of a reverse primer designed to amplify thebisulfite treated AHRR locus.

SEQ ID NO: 16: Sequence of a sequencing primer designed to sequence theamplicon produced from the bisulfite treated AHRR locus.

SEQ ID NO: 17: Sequence of the amplicon produced from the AHRR locusafter bisulfite treatment assuming 0% methylation of all CpG sites. Allcytosines, including cytosines from CpG sites are converted to thymines.

SEQ ID NO: 18: Sequence of the amplicon produced from the AHRR locusafter bisulfite treatment assuming 100% methylation of all CpG sites.Cytosines other than the cytosines from the CpG sites are converted tothymines. Cytosines from the CpG sites remain as cytosines.

SEQ ID NO: 19: Sequence of the SA-6 locus specific for buccal epithelialcell.

SEQ ID NO: 20: Sequence of a forward primer designed to amplify thebisulfite treated SA-6 locus.

SEQ ID NO: 21: Sequence of a reverse primer designed to amplify thebisulfite treated SA-6 locus.

SEQ ID NO: 22: Sequence of a sequencing primer designed to sequence theamplicon produced from the bisulfite treated SA-6 locus.

SEQ ID NO: 23: Sequence of the amplicon produced from the SA-6 locusafter bisulfite treatment assuming 0% methylation of all CpG sites. Allcytosines, including cytosines from CpG sites are converted to thymines.

SEQ ID NO: 24: Sequence of the amplicon produced from the SA-6 locusafter bisulfite treatment assuming 100% methylation of all CpG sites.Cytosines other than the cytosines from the CpG sites are converted tothymines. Cytosines from the CpG sites remain as cytosines.

DETAILED DESCRIPTION OF THE INVENTION

DNA methylation is one of the epigenetic mechanisms for gene regulation.Different DNA methylation levels in certain genetic loci control geneexpression by silencing or activating specific genes. The presence of amethyl group on the 5′ carbon of a cytosine belonging to thedinucleotide CG (CpG) is believed to prevent the binding of thetranscription machinery to the promoter of a gene.

DNA methylation levels at specific genetic loci are different forcertain cells from a body fluid when compared to other cells. Forexample, DNA methylation levels at specific genetic loci are differentamong the cells from blood, vaginal secretions, saliva, or semen.Accordingly, certain embodiments of the invention provide materials andmethods for detecting body fluids in a sample, for example, a forensicsample, based on DNA analyses of the cells present in the sample.

The nucleotide coordinates for genetic loci mentioned herein correspondto University of California Santa Cruz genome browser and Assembly hg19(UCSC Human Genome Browser GRCh37/hg19 Assembly).

Two genetic loci, namely NMUR2 and UBE2U, are provided that are specificfor sperm. The DNA methylation levels at these genetic loci in thegenomic DNA obtained from a sperm are significantly different from theDNA methylation levels at these genetic loci in the genomic DNA obtainedfrom the cells from other body fluids, such as blood, vaginalsecretions, saliva, and seminal fluid with no sperms. The assaysdeveloped using the NMUR2 and/or UBE2U genetic loci can be used incombination with pyrosequencing and/or HRM analysis to determine the DNAmethylation levels at these loci.

One marker, namely, SA-6, is provided that is specific for cells insaliva, particularly, buccal epithelial cells, and can be used toidentify buccal epithelial cells from the cells obtained from other bodyfluids, such as semen, vaginal secretions, and blood. Buccal epithelialcells show a significantly different DNA methylation level at the SA-6genetic locus when compared to the DNA methylation level at this geneticlocus in the genomic DNA of the cells obtained from other body fluids.

One marker, namely, AHRR, is provided that is specific for blood cellsand can be used to identify blood cells from the cells obtained fromother body fluids, such as semen, vaginal secretions, and saliva. Bloodcells show a significantly different DNA methylation level at the AHRRgenetic locus when compared to the DNA methylation level at this geneticlocus in the genomic DNA of the cells obtained from other body fluids.

In certain embodiments, identifying a sperm based on the methodsprovided herein is performed by determining the DNA methylation level atthe sperm specific genetic locus, the NMUR2 locus. Determining the DNAmethylation level at the NMUR2 locus can be performed through the use ofa specific primer pair that amplifies from the bisulfite-treated genomicDNA at the NMUR2 locus, which has the sequence of SEQ ID NO: 1 in thegenomic DNA not treated with bisulfite. An amplicon corresponding to theNMUR2 locus can be obtained by PCR using the bisulfite-treated genomicDNA as a template and a primer pair comprising SEQ ID NOs: 2 and 3.

The amplicon corresponding to the NMUR2 locus obtained by PCR can besequenced using a sequencing primer comprising SEQ ID NO: 4. A skilledartisan can design a sequencing primer other than SEQ ID NO: 4 tosequence the NMUR2 locus based on the sequences of SEQ ID NOs: 1, 5, and6 and such embodiments are within the purview of the invention.Alternately, a sequencing primer can be designed based on an adapterintroduced into the amplicon by incorporating the adapter into one ofthe forward and reverse primers used for the PCR amplification.

The sperm specific NMUR2 locus having the sequence of SEQ ID NO: 1 is alocus of 277 base pairs on chromosome 5 and occupies the chromosomallocation from chr5:151784131 to chr1:151784407. The genome coordinatesfor the NMUR2 locus are chr5:151784131-151784407. The NMUR2 locuscontains seven CpG sites that are hypomethylated in the genomes ofsperms compared to other cells, particularly, blood cells, vaginalepithelial cells, buccal epithelial cells, or seminal cells other thansperms. Particularly, cytosine residues located at the positionschr5:151784216 (position 86 of SEQ ID NO: 1), chr5:151784218 (position88 of SEQ ID NO: 1), chr5:151784223 (position 93 of SEQ ID NO: 1),chr5:151784240 (position 110 of SEQ ID NO: 1), chr5:151784245 (position115 of SEQ ID NO: 1), chr5:151784252 (cg17433294) (position 122 of SEQID NO: 1), and chr5:151784258 (position 128 of SEQ ID NO: 1) arehypomethylated in the NMUR2 genetic locus within the genome of a spermcompared to other cells, particularly, blood cells, vaginal epithelialcells, buccal epithelial cells or seminal cells other than sperms.

In another embodiment, identifying sperms based on the methods providedherein is performed by determining the DNA methylation level at a spermspecific locus, the UBE2U locus. Determining DNA methylation at theUBE2U locus can be performed using a specific primer pair that amplifiesfrom the bisulfite-treated genomic DNA at the UBE2U locus, which has thesequence of SEQ ID NO: 7. An amplicon corresponding to the UBE2U locuscan be obtained by PCR using bisulfite-treated genomic DNA as a templateand a primer pair comprising SEQ ID NOs: 8 and 9.

The amplicon corresponding to the UBE2U locus obtained by PCR can besequenced using a sequencing primer comprising SEQ ID NO: 10. A skilledartisan can design a sequencing primer other than SEQ ID NO: 10 tosequence the UBE2U locus based on the sequences of SEQ ID NOs: 7, 11,and 12 and such embodiments are within the purview of the invention.Alternately, a sequencing primer can be designed based on an adapterintroduced into the amplicon by incorporating the adapter into one ofthe forward and reverse primers used for the PCR amplification.

The sperm specific UBE2U locus having the sequence of SEQ ID NO: 7 is alocus of 207 base pairs on chromosome 1 and occupies the chromosomallocation from chr1:64669451 to chr1:64669657. The genome coordinates forthe UBE2U locus are chr1:64669451-64669657. The UBE2U locus containsthree CpG sites that are hypomethylated in the genomes of spermscompared to other cells, particularly, blood cells, vaginal epithelialcells, or buccal epithelial cells. Particularly, cytosine residueslocated at the positions chr1:64669473 (cg25108325) (position 23 of SEQID NO: 7), chr1:64669489 (position 39 of SEQ ID NO: 7), andchr1:64669492 (position 42 of SEQ ID NO: 7) are hypomethylated in thegenomes of sperms compared to other cells, particularly, blood cells,vaginal epithelial cells, buccal epithelial cells or seminal cells otherthan sperms.

In certain embodiments, identifying blood cells based on the methodsprovided herein is performed by determining the DNA methylation level ata blood cell specific locus, the AHRR locus. Determining DNA methylationat the AHRR locus can be performed through the use of a specific primerpair that amplifies from the bisulfite-treated genomic DNA at the AHRRlocus, which has the sequence of SEQ ID NO: 13. An ampliconcorresponding to the AHRR locus can be obtained by PCR usingbisulfate-treated genomic DNA as template and a primer pair comprisingSEQ ID NOs: 14 and 15.

The amplicon corresponding to the AHRR locus obtained by PCR can besequenced using a sequencing primer comprising SEQ ID NO: 16. A skilledartisan can design a sequencing primer other than SEQ ID NO: 16 tosequence the AHRR locus based on the sequences of SEQ ID NOs: 13, 17,and 18 and such embodiments are within the purview of the invention.Alternately, a sequencing primer can be designed based on an adapterintroduced into the amplicon by incorporating the adapter into one ofthe forward and reverse primers used for the PCR amplification.

The blood cell specific AHRR locus having the sequence of SEQ ID NO: 13is a locus of 233 base pairs on chromosome 5 and occupies thechromosomal location from chr5:368763 to chr5:368995. The genomecoordinates for the AHRR locus are chr5:368763-chr5:368995. The AHRRlocus contains four CpG sites that are hypomethylated in the genome of ablood cell compared to other cells, particularly, sperms, vaginalepithelial cells, buccal epithelial cells, or seminal cells other thansperms. Particularly, cytosine residues located at the positionschr5:368800 (position 38 of SEQ ID NO: 13), chr5:368804 (position 42 ofSEQ ID NO: 13), chr5:368821 (position 59 of SEQ ID NO: 13), chr5:368843(cg11902777) (position 81 of SEQ ID NO: 13) are hypomethylated in thegenome of a blood cell compared to other cells, particularly, sperms,vaginal epithelial cells, buccal epithelial cells or seminal cells otherthan sperms.

In certain embodiments, identifying buccal epithelial cells based on themethods provided herein is performed by determining the DNA methylationlevel at a buccal epithelial cell specific locus, the SA-6 locus.Determining DNA methylation at the SA-6 locus can be performed throughthe use of a specific primer pair that amplifies from thebisulfite-treated genomic DNA at the SA-6 locus, which has the sequenceof SEQ ID NO: 19. An amplicon corresponding to the SA-6 locus can beobtained by PCR using bisulfite-treated genomic DNA as template and aprimer pair comprising SEQ ID NOs: 20 and 21.

The amplicon corresponding to the SA-6 locus obtained by PCR can besequenced using a sequencing primer comprising SEQ ID NO: 22. A skilledartisan can design a sequencing primer other than SEQ ID NO: 22 tosequence the SA-6 locus based on the sequences of SEQ ID NOs: 19, 23 and24 and such embodiments are within the purview of the invention.Alternately, a sequencing primer can be designed based on an adapterintroduced into the amplicon by incorporating the adapter into one ofthe forward and reverse primers used for the PCR amplification.

The buccal epithelial cell specific SA-6 locus having the sequence ofSEQ ID NO: 19 is a locus of 124 base pairs on chromosome 2 and occupiesthe chromosomal location from chr2:63285615-chr2:63285738. The genomecoordinates for the SA-6 locus are chr2:63285615-chr2:63285738. The SA-6locus contains three CpG sites that are hypermethylated in the genome ofa buccal epithelial cell compared to other cells, particularly, sperms,vaginal epithelial cells, blood cells, or seminal cells other thansperms. Particularly, cytosine residues located at the positionschr2:63285645 (position 31 of SEQ ID NO: 19), chr2:63285654 (position 40of SEQ ID NO: 13), and chr2:63285682 (position 68 of SEQ ID NO: 19), arehypermethylated in the genome of a buccal epithelial cell compared toother cells, particularly, sperms, vaginal epithelial cells, bloodcells, or seminal cells other than sperms.

The methods described herein can be practiced with minute amounts ofgenomic DNA, for example, between 1 ng to 50 ng, particularly, between 5ng to 30 ng, more particularly, about 20 ng. Moreover, determining theDNA methylation levels at the specific genetic loci described herein,for example, SEQ ID NOs: 1, 7, 13, and 19, can be performed whenmixtures of body fluids are present.

Accordingly, one embodiment of the invention provides a method foridentifying a sample as containing or not containing a sperm, a buccalepithelial cell, or a blood cell, the method comprising the steps of:

a) determining the DNA methylation levels at the genetic locicorresponding to one or more of SEQ ID NOs: 1, 7, 13, and 19, in:

-   -   i) a genomic DNA isolated from the sample, and    -   ii) optionally, a control genomic DNA;

b) optionally, obtaining one or more reference values corresponding tothe DNA methylation levels at the genetic loci corresponding to one ormore of SEQ ID NOs: 1, 7, 13, and 19; and

c) identifying the sample as:

-   -   i) containing or not containing the sperm based on the DNA        methylation level at the genetic locus comprising SEQ ID NO: 1        in the genomic DNA isolated from the sample,    -   ii) containing or not containing the sperm based on the DNA        methylation level at the genetic locus comprising SEQ ID NO: 7        in the genomic DNA isolated from the sample,    -   iii) containing or not containing the blood cell based on the        DNA methylation level at the genetic locus comprising SEQ ID NO:        13 in the genomic DNA isolated from the sample, and/or    -   iv) containing or not containing the buccal epithelial cell        based on the DNA methylation level at the genetic locus        comprising SEQ ID NO: 19 in the genomic DNA isolated from the        sample.

In one embodiment, the invention provides a method for identifying asample as containing or not containing, a sperm, the method comprisingthe steps of:

a) determining the DNA methylation level at the genetic locus comprisingSEQ ID NO: 1 in:

-   -   i) a genomic DNA isolated from the sample, and    -   ii) optionally, a control genomic DNA;

b) optionally, obtaining one or more reference values corresponding tothe DNA methylation level at the genetic locus comprising SEQ ID NO: 1;and

c) identifying the sample as containing or not containing the spermbased on the DNA methylation level at the genetic locus comprising SEQID NO: 1 in the genomic DNA isolated from the sample.

In certain such embodiments, the methods further comprise identifying asample as containing or not containing a sperm, a buccal epithelialcell, or a blood cell, the method comprising the steps of:

a) determining the DNA methylation levels at the genetic locicorresponding to one or more of SEQ ID NOs: 7, 13, and 19, in:

-   -   i) a genomic DNA isolated from the sample, and    -   ii) optionally, a control genomic DNA;

b) optionally, obtaining one or more reference values corresponding tothe DNA methylation levels at the genetic loci corresponding to one ormore of SEQ ID NOs: 7, 13, and 19; and

c) identifying the sample as:

-   -   i) containing or not containing the sperm based on the DNA        methylation level at the genetic locus comprising SEQ ID NO: 7        in the genomic DNA isolated from the sample,    -   ii) containing or not containing the blood cell based on the DNA        methylation level at the genetic locus comprising SEQ ID NO: 13        in the genomic DNA isolated from the sample, and/or    -   iii) containing or not containing the buccal epithelial cell        based on the DNA methylation level at the genetic locus        comprising SEQ ID NO: 19 in the genomic DNA isolated from the        sample.

In another embodiment, the invention provides a method for identifying asample as containing or not containing a sperm, the method comprisingthe steps of:

a) determining the DNA methylation level at the genetic locus comprisingSEQ ID NO: 7 in:

-   -   i) a genomic DNA isolated from the sample, and    -   ii) optionally, a control genomic DNA;

b) optionally, obtaining one or more reference values corresponding tothe DNA methylation level at the genetic locus comprising SEQ ID NO: 7;and

c) identifying the sample as containing or not containing the spermbased on the DNA methylation level at the genetic locus comprising SEQID NO: 7 in the genomic DNA isolated from the sample.

In certain such embodiments, the methods further comprise identifying asample as containing or not containing one or more of a sperm, a buccalepithelial cell, and a blood cell, the method comprising the steps of:

a) determining the DNA methylation levels at the genetic locicorresponding to one or more of SEQ ID NOs: 1, 13, and 19, in:

-   -   i) a genomic DNA isolated from the sample, and    -   ii) optionally, a control genomic DNA;

b) optionally, obtaining one or more reference values corresponding tothe DNA methylation levels at the genetic loci corresponding to one ormore of SEQ ID NOs: 1, 13, and 19; and

c) identifying the sample as:

-   -   i) containing or not containing the sperm based on the DNA        methylation level at the genetic locus comprising SEQ ID NO: 1        in the genomic DNA isolated from the sample,    -   ii) containing or not containing the blood cell based on the DNA        methylation level at the genetic locus comprising SEQ ID NO: 13        in the genomic DNA isolated from the sample, and/or    -   iii) containing or not containing the buccal epithelial cell        based on the DNA methylation level at the genetic locus        comprising SEQ ID NO: 19 in the genomic DNA isolated from the        sample.

In a further embodiment, the invention provides a method for identifyinga sample as containing or not containing a blood cell, the methodcomprising the steps of:

a) determining the DNA methylation level at the genetic locus comprisingSEQ ID NO: 13 in:

-   -   i) a genomic DNA isolated from the sample, and    -   ii) optionally, a control genomic DNA;

b) optionally, obtaining one or more reference values corresponding tothe DNA methylation level at the genetic locus comprising SEQ ID NO: 13;and

c) identifying the sample as containing or not containing the blood cellbased on the DNA methylation level at the genetic locus comprising SEQID NO: 13 in the genomic DNA isolated from the sample.

In certain such embodiments, the methods further comprise identifying asample as containing or not containing a sperm or a buccal epithelialcell, the method comprising the steps of:

a) determining the DNA methylation levels at the genetic locicorresponding to one or more of SEQ ID NOs: 1, 7, and 19, in:

-   -   i) a genomic DNA isolated from the sample, and    -   ii) optionally, a control genomic DNA;

b) optionally, obtaining one or more reference values corresponding tothe DNA methylation levels at the genetic loci corresponding to one ormore of SEQ ID NOs: 1, 7, and 19; and

c) identifying the sample as:

-   -   i) containing or not containing the sperm based on the DNA        methylation level at the genetic locus comprising SEQ ID NO: 1        in the genomic DNA isolated from the sample,    -   ii) containing or not containing the sperm based on the DNA        methylation level at the genetic locus comprising SEQ ID NO: 7        in the genomic DNA isolated from the sample, and/or    -   iii) containing or not containing the buccal epithelial cell        based on the DNA methylation level at the genetic locus        comprising SEQ ID NO: 19 in the genomic DNA isolated from the        sample.

In an even further embodiment, the invention provides a method foridentifying a sample as containing or not containing a buccal epithelialcell, the method comprising the steps of:

a) determining the DNA methylation level at the genetic locus comprisingSEQ ID NO: 19 in:

-   -   i) a genomic DNA isolated from the sample, and    -   ii) optionally, a control genomic DNA;

b) optionally, obtaining one or more reference values corresponding tothe DNA methylation level at the genetic locus comprising SEQ ID NO: 19;and

c) identifying the sample as containing or not containing the buccalepithelial cell based on the DNA methylation level at the genetic locuscomprising SEQ ID NO: 19 in the genomic DNA isolated from the sample.

In certain such embodiments, the methods further comprise identifying asample as containing or not containing a sperm or a blood cell, themethod comprising the steps of:

a) determining the DNA methylation levels at the genetic locicorresponding to one or more of SEQ ID NOs: 1, 7, and 13, in:

-   -   i) a genomic DNA isolated from the sample, and    -   ii) optionally, a control genomic DNA;

b) optionally, obtaining one or more reference values corresponding tothe DNA methylation levels at the genetic loci corresponding to one ormore of SEQ ID NOs: 1, 7, and 13; and

c) identifying the sample as:

-   -   i) containing or not containing the sperm based on the DNA        methylation level at the genetic locus comprising SEQ ID NO: 1        in the genomic DNA isolated from the sample,    -   ii) containing or not containing the sperm based on the DNA        methylation level at the genetic locus comprising SEQ ID NO: 7        in the genomic DNA isolated from the sample, and/or    -   iii) containing or not containing the blood cell based on the        DNA methylation level at the genetic locus comprising SEQ ID NO:        13 in the genomic DNA isolated from the sample.

The control sample used in the methods of the invention can be obtainedfrom one or more of the following: a known vaginal epithelial cell, aknown sperm, a known blood cell, a known seminal cell other than sperm,and a known buccal epithelial cell. The control sample can also be acell other than a vaginal epithelial cell, sperm, blood cell, or buccalepithelial cell and that is known to have the DNA methylation level atthe specific genetic loci corresponding to SEQ ID NO: 1 or 7 to bedifferent from the DNA methylation level at the corresponding geneticlocus in a sperm cell. The control sample can also be a cell other thana vaginal epithelial cell, sperm, blood cell, or buccal epithelial celland that is known to have the DNA methylation level at the specificgenetic locus corresponding to SEQ ID NO: 13 to be different from theDNA methylation level at the corresponding genetic locus in a bloodcell. The control sample can also be a cell other than a vaginalepithelial cell, sperm, blood cell, or buccal epithelial cell and thatis known to have the DNA methylation level at the specific genetic locuscorresponding to SEQ ID NO: 19 to be different from the DNA methylationlevel at the corresponding genetic locus in a buccal epithelial cell.

If the control sample is a sperm, the step of identifying the sample ascontaining the sperm is based on the DNA methylation level at thegenetic locus comprising SEQ ID NO: 1 or 7 in the genomic DNA isolatedfrom the sample being similar to the DNA methylation level at thegenetic locus comprising SEQ ID NO: 1 or 7 in the control genomic DNA.Also, if the control sample is a sperm, the step of identifying thesample as not containing the sperm is based on the DNA methylation levelat the genetic locus comprising SEQ ID NO: 1 or 7 in the genomic DNAisolated from the sample being different from the DNA methylation levelat the genetic locus comprising SEQ ID NO: 1 or 7 in the control genomicDNA.

If the control sample is a cell different from a sperm, the step ofidentifying the sample as containing the sperm is based on the DNAmethylation level at the genetic locus comprising SEQ ID NO: 1 or 7 inthe genomic DNA isolated from the sample being different from the DNAmethylation level at the genetic locus comprising SEQ ID NO: 1 or 7 inthe control genomic DNA. Also, if the control sample is a cell differentfrom a sperm, the step of identifying the sample as not containing thesperm is based on the DNA methylation level at the genetic locuscomprising SEQ ID NO: 1 or 7 in the genomic DNA isolated from the samplebeing similar to the DNA methylation level at the genetic locuscomprising SEQ ID NO: 1 or 7 in the control genomic DNA.

The reference value corresponding to the DNA methylation level at thegenetic locus comprising SEQ ID NO: 1 or 7 can indicate the DNAmethylation level at the genetic locus comprising SEQ ID NO: 1 or 7 in asperm or a cell other than a sperm. As such, the reference valuecorresponding to the DNA methylation level at the genetic locuscomprising SEQ ID NO: 1 or 7 can indicate the presence or absence of asperm. In a sperm, the NMUR2 genetic locus is methylated at less thanabout 20%, less than about 15%, less than about 10%, less than about 5%,or less than about 1%. Also, in a sperm, the UBE2U genetic locus ismethylated at less than about 20%, less than about 15%, less than about10%, less than about 5%, or less than about 1%.

Similarly, if the control sample is a blood cell, the step ofidentifying the sample as containing the blood cell is based on the DNAmethylation level at the genetic locus comprising SEQ ID NO: 13 in thegenomic DNA isolated from the sample being similar to the DNAmethylation level at the genetic locus comprising SEQ ID NO: 13 in thecontrol genomic DNA. Also, if the control sample is a blood cell, thestep of identifying the sample as not containing the blood cell is basedon the DNA methylation level at the genetic locus comprising SEQ ID NO:13 in the genomic DNA isolated from the sample being different from theDNA methylation level at the genetic locus comprising SEQ ID NO: 13 inthe control genomic DNA.

If the control sample is a cell different from a blood cell, the step ofidentifying the sample as containing the blood cell is based on the DNAmethylation level at the genetic locus comprising SEQ ID NO: 13 in thegenomic DNA isolated from the sample being different from the DNAmethylation level at the genetic locus comprising SEQ ID NO: 13 in thecontrol genomic DNA. Also, if the control sample is a cell differentfrom a blood cell, the step of identifying the sample as not containingthe blood cell is based on the DNA methylation level at the geneticlocus comprising SEQ ID NO: 13 in the genomic DNA isolated from thesample being similar to the DNA methylation level at the genetic locuscomprising SEQ ID NO: 13 in the control genomic DNA.

The reference value corresponding to the DNA methylation level at thegenetic locus comprising SEQ ID NO: 13 can indicate the DNA methylationlevel at the genetic locus comprising SEQ ID NO: 13 in a blood cell or acell other than a blood cell. As such, the reference value correspondingto the DNA methylation level at the genetic locus comprising SEQ ID NO:13 can indicate the presence or absence of a blood cell. In a bloodcell, the AHRR genetic locus is methylated at less than about 20%, lessthan about 15%, less than about 10%, less than about 5%, or less thanabout 1%.

Further, if the control sample is a buccal epithelial cell, the step ofidentifying the sample as containing the buccal epithelial cell is basedon the DNA methylation level at the genetic locus comprising SEQ ID NO:19 in the genomic DNA isolated from the sample being similar to the DNAmethylation level at the genetic locus comprising SEQ ID NO: 19 in thecontrol genomic DNA. Also, if the control sample is a buccal epithelialcell, the step of identifying the sample as not containing the buccalepithelial cell is based on the DNA methylation level at the geneticlocus comprising SEQ ID NO: 19 in the genomic DNA isolated from thesample being different from the DNA methylation level at the geneticlocus comprising SEQ ID NO: 19 in the control genomic DNA.

If the control sample is a cell different from a buccal epithelial cell,the step of identifying the sample as containing the buccal epithelialcell is based on the DNA methylation level at the genetic locuscomprising SEQ ID NO: 19 in the genomic DNA isolated from the samplebeing different from the DNA methylation level at the genetic locuscomprising SEQ ID NO: 19 in the control genomic DNA. Also, if thecontrol sample is a cell different from a buccal epithelial cell, thestep of identifying the sample as not containing the buccal epithelialcell is based on the DNA methylation level at the genetic locuscomprising SEQ ID NO: 19 in the genomic DNA isolated from the samplebeing similar to the DNA methylation level at the genetic locuscomprising SEQ ID NO: 19 in the control genomic DNA.

The reference value corresponding to the DNA methylation level at thegenetic locus comprising SEQ ID NO: 19 can indicate the DNA methylationlevel at the genetic locus comprising SEQ ID NO: 19 in a buccalepithelial cell or a cell other than a buccal epithelial cell. As such,the reference value corresponding to the DNA methylation level at thegenetic locus comprising SEQ ID NO: 19 can indicate the presence orabsence of a buccal epithelial cell. In a buccal epithelial cell, theSA-6 genetic locus is methylated at more than about 60%, more than about65%, more than about 70%, more than about 80%, or more than about 85%.

In one embodiment, the DNA methylation level of specific genetic lociare used to determine the purity of a cell sample comprising orconsisting of one or more of a vaginal epithelial cell, sperm, bloodcell, and buccal epithelial cell.

For example, if a sperm sample is obtained, the DNA methylation levelsat the NMUR2 and/or the UBE2U loci can be checked in the sample toidentify the purity of the sperm sample. For example, if the NMUR2 orthe UBE2U genetic locus is about 1-20% methylated, the sperm sample isalmost 100% pure; whereas, if the NMUR2 or UBE2U genetic locus is onlyabout 50% methylated, the sperm sample is not pure and may contain atleast 30% of other types of cells, particularly, vaginal epithelialcells, blood cells, or buccal epithelial cells.

Similarly, if a blood cell sample is obtained, the DNA methylation levelat the AHRR locus can be checked in the sample to identify the purity ofthe blood cell sample. For example, if the AHRR genetic locus is about1-20% methylated, the blood cell sample is almost 100% pure; whereas, ifthe AHRR genetic locus is only about 50% methylated, the blood cellsample is not pure and may contain at least 30% other types of cells,particularly, vaginal epithelial cells, sperms, or buccal epithelialcells.

Moreover, if a buccal epithelial cell sample is obtained, the DNAmethylation level at the SA-6 locus can be checked in the sample toidentify the purity of the buccal epithelial cell sample. For example,if the SA-6 genetic locus is at least about 60-80% methylated, thebuccal epithelial cell sample is almost 100% pure; whereas, if the SA-6genetic locus is only about 30-40% methylated, the buccal epithelialcell sample is not pure and may contain at least 30% of other types ofcells, particularly, vaginal epithelial cells, sperms, or blood cells.

Various techniques are known to a person of ordinary skill in the art todetermine the DNA methylation level at the specific loci in a genomicDNA. Non-limiting examples of such techniques include bisulfiteconversion, HRM analysis, digestion by restriction enzymes followed byPCR, Combined Bisulfite Restriction Analysis (COBRA), direct sequencing,cloning and sequencing, bisulfite treatment and sequencing, bisulfitetreatment and pyrosequencing, mass spectrometry analysis, andprobe/microarray based assay. Certain techniques of determiningmethylation at certain genomic sites are described in Eads et al.,Combined bisulfite restriction analysis (COBRA), Methods MolecularBiology, 2002; 200:71-85; Xiong Z et al., COBRA: a sensitive andquantitative DNA methylation assay, Nucleic Acids Research, 1997,25:2532-4; Paul et al., Cytosine methylation: quantitation by automatedgenomic sequencing and GENESCAN analysis, Biotechniques, 1996,21:126-33; Warnecke et al., Identification and resolution of artifactsin bisulfite sequencing, Methods, 2002, 27:101-7; Tost et al., Analysisof gene-specific DNA methylation patterns by pyrosequencing technology,Methods Molecular Biology, 2007, 373:89-102; and Ehrich et al.,Quantitative high-throughput analysis of DNA methylation patterns bybase-specific cleavage and mass spectrometry, Proceedings of theNational Academy of Sciences, 2005; 102:15785-90. Each of thesereferences is herein incorporated by reference its entirety. Additionaltechniques for determining the DNA methylation level at a genetic locusare known to a person of ordinary skill in the art and such techniquesare within the purview of the invention.

In a particular embodiment, the DNA methylation level at the specificlocus in a genomic DNA obtained from a sample is determined by HRManalysis. HRM analysis involves bisulfite treatment of genomic DNA.Bisulfite treatment of genomic DNA chemically changes the unmethylatedcytosines to uracil, while the methylated cytosines are unchanged. Inthe PCR reaction using primers designed to amplify a specific locus,uracils are copied as thymines making the PCR product produced from anun-methylated locus thymine-rich, thereby reducing the meltingtemperature (Tm) of the amplicon compared to the PCR product producedfrom a methylated locus, which is cytosine-rich. As such, methylation ofthe specific locus can be determined based on Tm of the amplicon, i.e.,higher Tm indicates a methylated locus and lower Tm indicates anun-methylated locus. This technique is referred to as FIRM analysis.

Determining the DNA methylation level at the specific locus in a genomicDNA obtained from a sample by HRM analysis comprises the steps of:obtaining the sample, isolating genomic DNA from the sample, treatingthe isolated DNA with bisulfite, PCR amplifying the genetic loci usingspecifically designed primers to produce amplicons corresponding to thegenetic loci, determining the melting temperatures of the ampliconsproduced in the PCR, determining the DNA methylation levels at thespecific loci in a genomic DNA based on the melting temperatures of theamplicons. The DNA methylation levels at the specific genetic loci canbe used to identify the source of body fluid based on the difference inthe melting temperatures of the amplicons produced from the methylatedand unmethylated genetic loci.

In one embodiment of the invention, amplification and melt analysis areperformed in a single instrument, namely, a real time PCR instrumentwith melt capacity. Use of a single instrument diminishes the hands-ontime making the methods efficient.

In one embodiment, a primer pair designed to amplify the genetic locusafter bisulfite treatment of genomic DNA comprising SEQ ID NO: 1comprises a forward primer comprising SEQ ID NO: 2 and a reverse primercomprising SEQ ID NO: 3. A skilled artisan can design primer pairs otherthan SEQ ID NOs: 2 and 3 to amplify the NMUR2 genetic locus based on thebisulfite treatment of sequence of SEQ ID NO: 1 and the sequences of theregions flanking the NMUR2 genetic locus in the genomic DNA. Suchembodiments are within the purview of the invention.

In another embodiment, a primer pair designed to amplify the geneticlocus after bisulfite treatment of genomic DNA comprising SEQ ID NO: 7comprises a forward primer comprising SEQ ID NO: 8 and a reverse primercomprising SEQ ID NO: 9. A skilled artisan can design primer pairs otherthan SEQ ID NOs: 8 and 9 to amplify the UBE2U genetic locus based on thebisulfite treatment of sequence of SEQ ID NO: 7 and the sequences of theregions flanking the UBE2U locus in the genomic DNA. Such embodimentsare within the purview of the invention.

In a further embodiment, a primer pair designed to amplify the geneticlocus comprising SEQ ID NO: 13 comprises a forward primer comprising SEQID NO: 14 and a reverse primer comprising SEQ ID NO: 15. A skilledartisan can design primer pairs other than SEQ ID NOs: 14 and 15 toamplify the AHRR genetic locus based on the sequence of SEQ ID NO: 13and the sequences of regions flanking the AHRR locus in the genomic DNA.Such embodiments are within the purview of the invention.

In another embodiment, a primer pair designed to amplify the geneticlocus after bisulfite treatment of genomic DNA comprising SEQ ID NO: 19comprises a forward primer comprising SEQ ID NO: 20 and a reverse primercomprising SEQ ID NO: 21. A skilled artisan can design primer pairsother than SEQ ID NOs: 20 and 21 to amplify the SA-6 genetic locus basedon the bisulfite treatment of sequence of SEQ ID NO: 19 and thesequences of the regions flanking the SA-6 locus in the genomic DNA.Such embodiments are within the purview of the invention.

Accordingly, in one embodiment, the invention provides a method foridentifying a sample as containing or not containing one or more humancells selected from a sperm, buccal epithelial cell, and a blood cell,the method comprising the steps of:

a) isolating genomic DNA from the sample and, optionally, a controlsample;

b) treating the isolated genomic DNA with bisulfite;

c) PCR amplifying the bisulfite treated genetic loci corresponding toone or more of SEQ ID NOs: 1, 7, 13, and 19 to produce the correspondingone or more amplicons, wherein the PCR amplifying is performed using oneor more primer pairs selected from:

-   -   i) SEQ ID NOs: 2 and 3,    -   ii) SEQ ID NOs: 8 and 9,    -   iii) SEQ ID NOs: 14 and 15, and    -   iv) SEQ ID NOs: 20 and 21; and

d) determining the melting temperatures of the one or more amplicons;and

e) identifying the sample as:

-   -   i) containing or not containing the sperm based on the melting        temperature of the amplicon corresponding to the bisulfite        treated genetic locus comprising SEQ ID NO: 1 in the genomic DNA        isolated from the sample,    -   ii) containing or not containing the sperm based on the melting        temperature of the amplicon corresponding to the bisulfite        treated genetic locus comprising SEQ ID NO: 7 in the genomic DNA        isolated from the sample,    -   iii) containing or not containing the blood cell based on the        melting temperature of the amplicon corresponding to the genetic        locus comprising SEQ ID NO: 13 in the genomic DNA isolated from        the sample, and/or    -   iv) containing or not containing the buccal epithelial cell        based on the melting temperature of the amplicon corresponding        to the bisulfite treated genetic locus comprising SEQ ID NO: 19        in the genomic DNA isolated from the sample.

In a specific embodiment, the invention provides a method foridentifying a sample as containing or not containing a sperm based onthe DNA methylation level of SEQ ID NO: 1 determined using a primer paircomprising SEQ ID NOs: 2 and 3. Such methods can further compriseidentifying a sample as containing or not containing a sperm, a bloodcell, or a buccal epithelial cell based on the DNA methylation levels ofone or more of SEQ ID NOs: 7, 13, and 19 determined using a primer paircomprising SEQ ID NOs: 8 and 9, a primer pair comprising SEQ ID NOs: 14and 15, and a primer pair comprising SEQ ID NOs: 20 and 21,respectively.

In another embodiment, the invention provides a method for identifying asample as containing or not containing a sperm based on the DNAmethylation level of SEQ ID NO: 7 determined using a primer paircomprising SEQ ID NOs: 8 and 9. Such methods can further compriseidentifying a sample as containing or not containing a sperm, a bloodcell, or a buccal epithelial cell based on the DNA methylation levels ofone or more of SEQ ID NOs: 1, 13, and 19 determined using a primer paircomprising SEQ ID NOs: 2 and 3, a primer pair comprising SEQ ID NOs: 14and 15, and a primer pair comprising SEQ ID NOs: 20 and 21 respectively.

In a further embodiment, the invention provides a method for identifyinga sample as containing or not containing a blood cell based on the DNAmethylation level of SEQ ID NO: 13 determined using a primer paircomprising SEQ ID NOs: 14 and 15. Such methods can further compriseidentifying a sample as containing or not containing a sperm or a buccalepithelial cell based on the DNA methylation levels of one or both ofSEQ ID NOs: 1 and 7 determined using a primer pair comprising SEQ IDNOs: 2 and 3 and a primer pair comprising SEQ ID NOs: 8 and 9,respectively, and the DNA methylation levels of SEQ ID NO: 19 determinedusing a primer pair comprising SEQ ID NOs: 20 and 21.

In an even further embodiment, the invention provides a method foridentifying a sample as containing or not containing a buccal epithelialcell based on the DNA methylation level of SEQ ID NO: 19 determinedusing a primer pair comprising SEQ ID NOs: 20 and 21. Such methods canfurther comprise identifying a sample as containing or not containing asperm or a blood cell based on the DNA methylation levels of one or bothof SEQ ID NOs: 1 and 7 determined using a primer pair comprising SEQ IDNOs: 2 and 3 and a primer pair comprising SEQ ID NOs: 8 and 9,respectively, or the DNA methylation levels of SEQ ID NO: 13 determinedusing a primer pair comprising SEQ ID NOs: 14 and 15.

In one embodiment, the melting temperature of one or more ampliconscorresponding to the bisulfite treated genetic loci corresponding to SEQID NOs: 1, 7, or 19 is compared to a reference value to determine theDNA methylation level at the genetic loci corresponding to SEQ ID NOs:1, 7, or 19, which in turn is used to identify the sample as containingor not containing a sperm, or a buccal epithelial cell.

In one embodiment, the melting temperature of between 80° C. and 82° C.,particularly, about 80.9° C., for the amplicon corresponding to thebisulfite treated genetic locus comprising SEQ ID NO: 1 is used toidentify the presence of a sperm in the sample; whereas, the meltingtemperature of above 82° C. for the amplicon corresponding to thebisulfite treated genetic locus comprising SEQ ID NO: 1 is used toidentify the absence of a sperm in the sample and/or the presence ofother body fluids, namely, saliva, blood, semen without sperms, orvaginal secretion.

In another embodiment, the melting temperature of between 76° C. and 78°C., particularly, about 77.1° C., for the amplicon corresponding to thebisulfite treated genetic locus comprising SEQ ID NO: 7 is used toidentify the presence of a sperm in the sample; whereas, the meltingtemperature of above 78° C. for the amplicon corresponding to thebisulfite treated genetic locus comprising SEQ ID NO: 7 is used toidentify the absence of a sperm in the sample and/or the presence ofother body fluids, namely, saliva, blood, semen without sperms, orvaginal secretion.

In a further embodiment, the melting temperature of between 77° C. and79° C., particularly, about 77.6° C., for the amplicon corresponding tothe bisulfite treated genetic locus comprising SEQ ID NO: 19 is used toidentify the presence of a buccal epithelial cell in the sample;whereas, the melting temperature of below 77° C. for the ampliconcorresponding to the bisulfite treated genetic locus comprising SEQ IDNO: 19 is used to identify the absence of a buccal epithelial cell inthe sample and/or the presence of other body fluids, namely, blood,semen, or vaginal secretion.

The four primer sets described above would amplify genomic DNA isolatedfrom samples containing one or more of vaginal epithelial cells orvaginal secretions, sperms or semen, blood or blood cells, and buccalepithelial cell or saliva. The identification of different body fluidscan be made after amplification and further analysis, for example, byHRM analysis or sequencing analysis, such as pyrosequencing analysis.

The primer pairs amplify bisulfite treated genomic DNA regardless of itsDNA methylation level and would not amplify genomic DNA that is notbisulfite treated. Therefore, if the DNA sample is not appropriate forPCR amplification, for example, due to a low amount of DNA or presenceof impurities, the amplicons will not be produced.

A control DNA sample can be used and treated in the same manner as atest sample to ensure that the reagents are working properly. Therefore,if a test sample fails to produce amplicons, the sample can beidentified as a source of a problem for lack of amplification despitehaving working reagents. Likewise a negative control can be run where,for example, water replaces the genomic DNA to ensure that anyamplification is not due to unspecific amplification arising fromcontaminated reagents.

A control primer pair can also be used that would amplify genomic DNAthat is not bisulfite converted. The presence of an amplicon for thisprimer pair would tell a user that the bisulfite conversion was notsuccessful. Therefore, instead of having an amplification that did notwork for an unknown reason, a user would identify a failed bisulfiteconversion.

In one embodiment, an amplicon corresponding to a genetic locus isdistinguished from other amplicons based on a fluorophore attached tothe primer pair designed to amplify the genetic locus. Differentfluorophores attached to different primer pairs can be detected atdifferent wavelengths using fluorescence detectors.

In one embodiment, each amplicon is labelled with a fluorophore anddifferent fluorophores are matched with different melt curves. Bymatching fluorophores with melt curves, the presence of different bodyfluids in a sample containing a mixture of body fluids can be detected.

In another embodiment, each primer pair is labelled with a fluorophorethat is detected at a specific wavelength. As the multiplex PCRproceeds, the fluorophore in the primers is quenched by a nucleotidethat specifically binds to the DNA amplicon. Fluorescence decreases withthe increase of amplification for that specific locus.

An example of detecting different fluorophores for different primerpairs is provided in the Plexor® qPCR and RT-PCR from Promega, Inc.Certain details of Plexor® qPCR are provided in Technical Manual forPlexor® qPCR (Promega, Literature #TM262, Revised September 2009), thecontents of which are herein incorporated by reference in its entirety.

In certain embodiments, a control genomic DNA can comprise DNA that isnot bisulfite treated. Therefore, each locus has a “control primer set”directed to a bisulfite untreated genomic DNA sequence and a “testprimer sequence” directed to bisulfite treated genomic DNA. Therefore,for every test, one can have a multiplex primer set directed to abisulfite treated test genomic DNA and a multiplex primer set directedto a bisulfite untreated control genomic DNA.

In a particular embodiment, the DNA methylation level at the specificlocus in a genomic DNA obtained from a sample is determined bysequencing, for example, pyrosequencing. Determining methylation of agenetic locus based on sequencing involves bisulfite treating a genomicDNA. Bisulfite treatment of genomic DNA chemically changes theunmethylated cytosines to uracil, while the methylated cytosines areunchanged. In the PCR reaction using primers designed to amplify aspecific bisulfite treated locus, uracils are copied as thymines in anun-methylated site and cytosines are copied as guanines in a methylatedsite. As such, methylation of the specific site can be determined basedon the presence of cytosine in the amplicon; whereas, lack ofmethylation can be determined based on the presence of cytosine in theamplicon.

Using a sequencing based method, such as pyrosequencing, determining theDNA methylation level at the specific locus in a genomic DNA obtainedfrom a sample by sequencing comprises the steps of: obtaining thesample, isolating genomic DNA from the sample, treating the isolated DNAwith bisulfite, PCR amplifying the genetic locus using specificallydesigned primers to produce an amplicon corresponding to the geneticlocus, determining the sequences of the amplicon produced in the PCR,and determining the DNA methylation level at the specific locus in agenomic DNA based on the sequence of the amplicon. The methylation levelfor a particular CpG site can be determine based on the ratio of C/T atthe CpG site. The DNA methylation level at the specific genetic locuscan be used to identify the source of body fluid.

Accordingly, in one embodiment, the invention provides a method foridentifying a sample as containing or not containing a sperm, a buccalepithelial cell, or a blood cell, the method comprising the steps of:

a) isolating genomic DNA from the sample and, optionally, a controlsample;

b) treating the isolated genomic DNA with bisulfite;

c) PCR amplifying the genetic loci corresponding to one or more of SEQID NOs: 1, 7, 13, and 19 to produce the corresponding one or moreamplicons, wherein the PCR amplifying is performed using one or moreprimer pairs selected from:

-   -   i) SEQ ID NOs: 2 and 3,    -   ii) SEQ ID NOs: 8 and 9,    -   iii) SEQ ID NOs: 14 and 15, or    -   iv) SEQ ID NOs: 20 and 21; and

d) determining the sequences of the one or more amplicons; and

e) identifying the sample as:

-   -   i) containing or not containing the sperm based on the sequence        of the amplicon corresponding to the bisulfite treated genetic        locus comprising SEQ ID NO: 1 in the genomic DNA isolated from        the sample,    -   ii) containing or not containing the sperm based on the sequence        of the amplicon corresponding to the bisulfite treated genetic        locus comprising SEQ ID NO: 7 in the genomic DNA isolated from        the sample,    -   iii) containing or not containing the blood cell based on the        sequence of the amplicon corresponding to the bisulfite treated        genetic locus comprising SEQ ID NO: 13 in the genomic DNA        isolated from the sample, and/or    -   iv) containing or not containing the buccal epithelial cell        based on the sequence of the amplicon corresponding to the        bisulfite treated genetic locus comprising SEQ ID NO: 19 in the        genomic DNA isolated from the sample.

The sequencing primer for sequencing the amplicon corresponding tobisulfite treated SEQ ID NO: 1, for example, an amplicon produced in aPCR using a primer pair comprising SEQ ID NOs: 2 and 3, can comprise SEQID NO: 4. Accordingly, the step of determining the sequences of theamplicon corresponding to bisulfite treated SEQ ID NO: 1 comprises usingthe sequencing primer comprising SEQ ID NO: 4. A skilled artisan candesign a sequencing primer other than SEQ ID NO: 4 to sequence the NMUR2locus based on the sequences of SEQ ID NOs: 1, 5, and 6 and suchembodiments are within the purview of the invention. Alternately, asequencing primer can be designed based on an adapter introduced intothe amplicon by incorporating the adapter into one of the forward andreverse primers.

The sequencing primer for sequencing the amplicon corresponding tobisulfite treated SEQ ID NO: 7, for example, an amplicon produced in aPCR using a primer pair comprising SEQ ID NOs: 8 and 9, can comprise SEQID NO: 10. Accordingly, the step of determining the sequences of theamplicon corresponding to bisulfite treated SEQ ID NO: 7 comprises usingthe sequencing primer comprising SEQ ID NO: 10. A skilled artisan candesign a sequencing primer other than SEQ ID NO: 10 to sequence theUBE2U locus based on the sequences of SEQ ID NOs: 7, 11, and 12 and suchembodiments are within the purview of the invention. Alternately, asequencing primer can be designed based on an adapter introduced intothe amplicon by incorporating the adapter into one of the forward andreverse primers.

The sequencing primer for sequencing the amplicon corresponding tobisulfite treated SEQ ID NO: 13, for example, an amplicon produced in aPCR using a primer pair comprising SEQ ID NOs: 14 and 15, can compriseSEQ ID NO: 16. Accordingly, the step of determining the sequences of theamplicon corresponding to bisulfite treated SEQ ID NO: 13 comprisesusing the sequencing primer comprising SEQ ID NO: 16. A skilled artisancan design a sequencing primer other than SEQ ID NO: 16 to sequence theAHRR locus based on the sequences of SEQ ID NOs: 13, 17, and 18 and suchembodiments are within the purview of the invention. Alternately, asequencing primer can be designed based on an adapter introduced intothe amplicon by incorporating the adapter into one of the forward andreverse primers.

The sequencing primer for sequencing the amplicon corresponding tobisulfite treated SEQ ID NO: 19, for example, an amplicon produced in aPCR using a primer pair comprising SEQ ID NOs: 20 and 21, can compriseSEQ ID NO: 22. Accordingly, the step of determining the sequences of theamplicon corresponding to bisulfite treated SEQ ID NO: 19 comprisesusing the sequencing primer comprising SEQ ID NO: 22. A skilled artisancan design a sequencing primer other than SEQ ID NO: 22 to sequence theSA-6 locus based on the sequences of SEQ ID NOs: 19, 23, and 24 and suchembodiments are within the purview of the invention. Alternately, asequencing primer can be designed based on an adapter introduced intothe amplicon by incorporating the adapter into one of the forward andreverse primers.

In one embodiment, the sequence of one or more amplicons correspondingto the bisulfite treated genetic loci corresponding to SEQ ID NOs: 1, 7,13, and 19 are compared to reference sequences to determine the DNAmethylation levels at the genetic loci comprising SEQ ID NOs: 1, 7, 13,and 19, which in turn is used to identify the sample as containing ornot containing a sperm, a buccal epithelial cell, or a blood cell.

A skilled artisan can design a sequencing primer to sequence theamplicons corresponding to the bisulfite treated genetic locuscomprising SEQ ID NOs: 1, 7, 13, or 19 based on the sequences of thesegenetic loci and the sequences of the regions flanking these geneticloci in the genomic DNA. Such embodiments are within the purview of theinvention.

In another embodiment, a sequencing primer can be designed based on anadapter introduced into the amplicon by incorporating the adapter intoone of the forward and reverse primers.

An “adapter” as used herein is a sequence of about 10 to 20 nucleotidesthat can be introduced into an amplicon by incorporating the adapterinto the primer used for the amplification of the amplicon. Once anamplicon contains an adapter sequence, a primer designed based on thesequence of the adapter can be used to sequence the amplicon.

In certain embodiments, the methods described herein to identify asample as containing or not containing a sperm, a buccal epithelialcell, and/or a blood cell are practiced on a forensic sample to detectthe presence of one or more of these cells in the forensic sample. Themethods can be practiced on a forensic sample that is processed toseparate a cell suspected to be a vaginal epithelial cell, sperm, bloodcell, or buccal epithelial cell before the step of isolating andanalyzing the genomic DNA. The methods can also be practiced on aforensic sample that is known to contain only vaginal epithelial cells,sperms, semen, blood cells, or buccal epithelial cells or a combinationthereof.

Certain embodiments of the invention also provide a method fordetermining the DNA methylation levels at the genetic loci correspondingto one or more of SEQ ID NOs: 1, 7, 13, and 19, in a genomic DNA from acell, the method comprising the steps of:

-   -   a) isolating the genomic DNA from the cell,    -   b) treating the genomic DNA with bisulfite,    -   c) conducting a PCR using the bisulfite treated genomic DNA as a        template and one or more primer pairs designed to produce        amplicons corresponding to the one or more of the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 7, 13, and 19,        and    -   d) analyzing the PCR amplicons produced in step c) to determine        the DNA methylation levels at the genetic loci corresponding to        one or more of SEQ ID NOs: 1, 7, 13, and 19.

In a specific embodiment, the invention provides a method fordetermining the DNA methylation level at the genetic locus comprisingSEQ ID NO: 1 in a genomic DNA from a cell, the method comprising thesteps of:

-   -   a) isolating the genomic DNA from the cell,    -   b) treating the genomic DNA with bisulfite,    -   c) conducting a PCR using the bisulfite treated genomic DNA as a        template and one or more primer pairs designed to produce        amplicons corresponding to the genetic locus corresponding SEQ        ID NO: 1, and    -   d) analyzing the PCR amplicon produced in step c) to determine        the DNA methylation level at the genetic locus comprising SEQ ID        NO: 1.

Certain such embodiments further comprise determining the DNAmethylation levels at genetic loci corresponding to one or more of SEQID NOs: 7, 13, and 19, in a genomic DNA from a cell, the methodcomprising the steps of:

-   -   a) isolating the genomic DNA from the cell,    -   b) treating the genomic DNA with bisulfite,    -   c) conducting a PCR using the bisulfite treated genomic DNA as a        template and one or more primer pairs designed to produce        amplicons corresponding to the one or more of the genetic loci        corresponding to SEQ ID NOs: 7, 13, and 19, and    -   d) analyzing the PCR amplicons produced in step c) to determine        the DNA methylation levels at the genetic loci corresponding to        one or more of SEQ ID NOs: 7, 13, and 19.

In another embodiment, the invention provides a method for determiningthe DNA methylation level at the genetic locus comprising SEQ ID NO: 7in a genomic DNA from a cell, the method comprising the steps of:

-   -   a) isolating the genomic DNA from the cell,    -   b) treating the genomic DNA with bisulfite,    -   c) conducting a PCR using the bisulfite treated genomic DNA as a        template and one or more primer pairs designed to produce        amplicons corresponding to the genetic locus corresponding SEQ        ID NO: 7, and    -   d) analyzing the PCR amplicon produced in step c) to determine        the DNA methylation level at the genetic locus comprising SEQ ID        NO: 7.

Certain such embodiments further comprise determining the DNAmethylation levels at genetic loci corresponding to one or more of SEQID NOs: 1, 13, and 19, in a genomic DNA from a cell, the methodcomprising the steps of:

-   -   a) isolating the genomic DNA from the cell,    -   b) treating the genomic DNA with bisulfite,    -   c) conducting a PCR using the bisulfite treated genomic DNA as a        template and one or more primer pairs designed to produce        amplicons corresponding to the one or more of the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 13, and 19, and    -   d) analyzing the PCR amplicons produced in step c) to determine        the DNA methylation levels at the genetic loci corresponding to        one or more of SEQ ID NOs: 1, 13, and 19.

In a further embodiment, the invention provides a method for determiningthe DNA methylation level at the genetic locus comprising SEQ ID NO: 13in a genomic DNA from a cell, the method comprising the steps of:

-   -   a) isolating the genomic DNA from the cell,    -   b) treating the genomic DNA with bisulfite,    -   c) conducting a PCR using the bisulfite treated genomic DNA as a        template and one or more primer pairs designed to produce        amplicons corresponding to the genetic locus corresponding SEQ        ID NO: 13, and    -   d) analyzing the PCR amplicon produced in step c) to determine        the DNA methylation level at the genetic locus comprising SEQ ID        NO: 13.

Certain such embodiments further comprise determining the DNAmethylation levels at genetic loci corresponding to one or more of SEQID NOs: 1, 7, and 19, in a genomic DNA from a cell, the methodcomprising the steps of:

-   -   a) isolating the genomic DNA from the cell,    -   b) treating the genomic DNA with bisulfite,    -   c) conducting a PCR using the bisulfite treated genomic DNA as a        template and one or more primer pairs designed to produce        amplicons corresponding to the one or more of the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 7, and 19, and    -   d) analyzing the PCR amplicons produced in step c) to determine        the DNA methylation levels at the genetic loci corresponding to        one or more of SEQ ID NOs: 1, 7, and 19.

In an even further embodiment, the invention provides a method fordetermining the DNA methylation level at the genetic locus comprisingSEQ ID NO: 19 in a genomic DNA from a cell, the method comprising thesteps of:

-   -   a) isolating the genomic DNA from the cell,    -   b) treating the genomic DNA with bisulfite,    -   c) conducting a PCR using the bisulfite treated genomic DNA as a        template and one or more primer pairs designed to produce        amplicons corresponding to the genetic locus corresponding SEQ        ID NO: 19, and    -   d) analyzing the PCR amplicon produced in step c) to determine        the DNA methylation level at the genetic locus comprising SEQ ID        NO: 19.

Certain such embodiments further comprise determining the DNAmethylation levels at genetic loci corresponding to one or more of SEQID NOs: 1, 7, and 13, in a genomic DNA from a cell, the methodcomprising the steps of:

-   -   a) isolating the genomic DNA from the cell,    -   b) treating the genomic DNA with bisulfite,    -   c) conducting a PCR using the bisulfite treated genomic DNA as a        template and one or more primer pairs designed to produce        amplicons corresponding to the one or more of the genetic loci        corresponding to one or more of SEQ ID NOs: 1, 7, and 13, and    -   d) analyzing the PCR amplicons produced in step c) to determine        the DNA methylation levels at the genetic loci corresponding to        one or more of SEQ ID NOs: 1, 7, and 13.

The DNA methylation levels at the one or more genetic loci comprisingSEQ ID NOs: 1, 7, 13, and 19 can be determined using HRM analysis orsequencing. The primer pairs and the sequencing primers provided abovein the methods of identifying a sample as containing or not containing asperm, a buccal epithelial cell, or a blood cell can also be used in themethods disclosed herein for determining the DNA methylation levels atthe one or more genetic loci comprising SEQ ID NOs: 1, 7, 13, and 19 andsuch embodiments are within the purview of the instant invention.

A further embodiment of the invention provides a kit comprising one ormore primer pairs designed to amplify the genetic loci corresponding toone or more SEQ ID NOs: 1, 7, 13, and 19 in a bisulfite treated humangenomic DNA.

In one embodiment, the kit comprises one or more primer pairs selectedfrom:

i) a primer pair comprising SEQ ID NOs: 2 and 3,

-   -   ii) a primer pair comprising SEQ ID NOs: 8 and 9,    -   iii) a primer pair comprising SEQ ID NOs: 14 and 15, and    -   iv) a primer pair comprising SEQ ID NOs: 20 and 21.

A skilled artisan can design additional primer pairs to amplify thebisulfite treated genetic loci corresponding to one or more SEQ ID NOs:1, 7, 13, and 19 based on the sequences of these genetic loci and thesequences of flanking regions in the genomic DNA and such embodimentsare within the purview of the invention.

In further embodiments, the invention provides a kit comprising:

-   -   i) the primer pair comprising SEQ ID NOs: 2 and 3 and a        sequencing primer comprising SEQ ID NO: 4,    -   ii) the primer pair comprising SEQ ID NOs: 8 and 9 and a        sequencing primer comprising SEQ ID NO: 10,    -   iii) the primer pair comprising SEQ ID NOs: 14 and 15 and a        sequencing primer comprising SEQ ID NO: 16, and/or    -   iv) the primer pair comprising SEQ ID NOs: 20 and 21 and a        sequencing primer comprising SEQ ID NO: 22.

A specific embodiment of the invention provides a kit comprising:

(a) a primer pair that amplifies in a PCR a DNA sequence consisting ofSEQ ID NO: 5 or 6, and optionally,

(b) a sequencing primer that sequences in a sequencing reaction anamplicon produced by a PCR conducted by using the primer pair thatamplifies the DNA sequence consisting of bisulfate treated SEQ ID NO: 1,

wherein each primer of the primer pair and, when present, the sequencingprimer, has a sequence complementary to the sequence of SEQ ID NO: 5 or6 and has between 15 and 30 nucleotides.

In certain such embodiments, the primer pair comprises a forward primercomprising SEQ ID NO: 2 and a reverse primer comprising SEQ ID NO: 3and, when present, the sequencing primer comprises SEQ ID NO: 4.

A further specific embodiment of the invention provides a kitcomprising:

(a) a primer pair that amplifies in a PCR a DNA sequence consisting ofSEQ ID NO: 11 or 12, and optionally,

(b) a sequencing primer that sequences in a sequencing reaction anamplicon produced by a PCR conducted by using the primer pair thatamplifies the DNA sequence consisting of bisulfite treated SEQ ID NO: 7,

wherein each primer of the primer pair and, when present, the sequencingprimer, has a sequence complementary to the sequence of SEQ ID NO: 11 or12 and has between 15 and 30 nucleotides.

In certain such embodiments, the primer pair comprises a forward primercomprising SEQ ID NO: 8 and a reverse primer comprising SEQ ID NO: 9and, when present, the sequencing primer comprises SEQ ID NO: 10.

An even further specific embodiment of the invention provides a kitcomprising:

(a) a primer pair that amplifies in a PCR a DNA sequence consisting ofSEQ ID NO: 17 or 18, and optionally,

(b) a sequencing primer that sequences in a sequencing reaction anamplicon produced by a PCR conducted by using the primer pair thatamplifies the DNA sequence consisting of bisulfite treated SEQ ID NO:13,

wherein each primer of the primer pair and, when present, the sequencingprimer, has a sequence complementary to the sequence of SEQ ID NO: 17 or18 and has between 15 and 30 nucleotides.

In certain such embodiments, the primer pair comprises a forward primercomprising SEQ ID NO: 14 and a reverse primer comprising SEQ ID NO: 15and, when present, the sequencing primer comprises SEQ ID NO: 16.

A further embodiment of the invention provides a kit comprising:

(a) a primer pair that amplifies in a PCR a DNA sequence consisting ofSEQ ID NO: 23 or 24, and optionally,

(b) a sequencing primer that sequences in a sequencing reaction anamplicon produced by a PCR conducted by using the primer pair thatamplifies the DNA sequence consisting of bisulfite treated SEQ ID NO:19,

wherein each primer of the primer pair and, when present, the sequencingprimer, has a sequence complementary to the sequence of SEQ ID NO: 23 or24 and has between 15 and 30 nucleotides.

In certain such embodiments, the primer pair comprises a forward primercomprising SEQ ID NO: 20 and a reverse primer comprising SEQ ID NO: 21and, when present, the sequencing primer comprises SEQ ID NO: 22.

In further embodiments, the kit comprises one or more reagents, forexample, reagents for treating a sample, reagents for isolating cellsfrom the sample, reagents for isolating genomic DNA from the sample,reagents for bisulfite treating the genomic DNA, reagents for conductingPCR, reagents for conducting pyrosequencing and reagent for conductingHRM analysis.

As used herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Further, to the extent that the terms “including,”“includes,” “having,” “has,” “with,” or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”The transitional terms/phrases (and any grammatical variations thereof)“comprising,” “comprises,” “comprise,” include the phrases “consistingessentially of,” “consists essentially of,” “consisting,” and“consists.”

The phrases “consisting essentially of” or “consists essentially of”indicate that the claim encompasses embodiments containing the specifiedmaterials or steps and those that do not materially affect the basic andnovel characteristic(s) of the claim.

The term “about” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,i.e., the limitations of the measurement system. Typically, “about”indicates within a range of 0 to 10% of a given value. For example, whenterm “about” is used in the context of the number of nucleotides inoligonucleotides; these oligonucleotides contain the stated number ofnucleotides with a variation of 0-10% around the value (X±10%). In thecontext of melting temperatures where the term “about” is used, themelting temperatures are within 0.5° C. of the stated meltingtemperature.

As used herein, the phrase “DNA methylation level” as applied to agenetic locus refers to whether one or more cytosine residues present ina CpG have or do not have a methylation group. The DNA methylation levelrefers to the percentage of cells in a sample that do or do not have amethylation group on such cytosines. For example, if 50 cells in a poolof 100 cells contain methylated cytosines at a CpG site, the DNAmethylation level at the CpG site is 50%.

A primer pair is a pair of oligonucleotides, each having about 15 to 30nucleotides, and designed to amplify a specific locus from a templateDNA. Similarly, a sequencing primer is an oligonucleotide having about15 to 30 nucleotides and designed to sequence an amplicon. Guidelinesfor designing a primer pair to amplify a specific locus in a templateDNA and a sequencing primer to sequence an amplicon are well known inthe art.

A primer pair designed to amplify a target locus comprises a forwardprimer and a reverse primer, each having about 15 to 30 nucleotides, andeach having a sequence complementary to the sequence towards the end ofthe target genomic locus. Similarly, a sequencing primer designed tosequence an amplicon has about 15 to 30 nucleotides and has a sequencecomplementary to a sequence within the target amplicon to be sequenced.

A person of ordinary skill in the art can design a primer pair or asequencing primer based on the target genetic locus.

A singleplex PCR is a reaction where only one set of primers is used perreaction;

whereas, a multiplex reaction is one that uses multiple primer sets perPCR reaction.

Materials and Methods

Sample Collection:

Forensically relevant body fluid samples were collected from unrelatedvolunteers. 23 venous blood, 24 saliva (buccal swab), 22 vaginalsecretion, and 20 semen samples containing sperms were collected.Freshly ejaculated semen was collected in a plastic cup and then putonto sterile cotton swabs to dry. Venous blood, saliva, and vaginalsecretions were collected directly on sterile cotton swabs and allowedto dry at room temperature. All biological samples were acquired fromvolunteers.

Screening Strategies:

Blood, saliva, semen, and vaginal secretion (n=3 per sample type) wereexamined to screen candidates of CpG sites located in 12 genomic loci toidentify their tissue-specific differentially methylated regions(tDMRs). In this step, candidates of CpG sites were examined bypyrosequencing. The CpG sites that showed differences in theirmethylated profiles between various body fluids were selected forfurther DNA methylation analyses by pyrosequencing and HRM analysis. Inthis study, only four tDMRs were discovered including locations at theNMUR2, UBE2U, SA-6, and AHRR loci.

DNA Extraction and Bisulfite Conversion:

Dried swabs containing various body fluid samples were DNA extracted byeither the EZ1® DNA Investigator Kit on the BioRobot® EZ1 automatedpurification workstation (Qiagen) or by standard organic extractionusing phenol-chloroform-isoamyl alcohol (Thermo Fisher Scientific). Theextracted DNA was bisulfite-modified using the EpiTect® Fast DNABisulfite Kit (Qiagen), which can modify 1 ng to 2 μg of DNA, to convertthe unmethylated cytosine to uracil.

Pyrosequencing:

To determine the DNA methylation levels at the potential markers invarious body fluid samples, sequencing was carried out bypyrosequencing. First, specific PCR primers and sequencing primer weredesigned using PyroMark Assay Design 2.0 software (Qiagen Inc. CA) toamplify the bisulfite modified target regions. After screening more than100 CpG sites across 12 genetic loci, three loci were found to be tissuespecific. The three assays located at the NMUR2, UBE2U, SA-6, and AHRRgenes were designed to target different numbers of CpG sites (Table 1).The specific locations were next amplified with one member of each PCRprimer pair labeled with biotin to produce biotinylated PCR ampliconsneeded for the downstream pyrosequencing reaction. The target regionswere amplified in a singleplex fashion by utilizing the PyroMark® PCRkit (Qiagen) on the GeneAmp® PCR system 9700 (Applied Biosystems, FosterCity, Calif.). The PCR reaction was modified to utilize 15 μl reactionvolumes based on the total volume specified by the manufacturer'sprotocol. The PCR products were pyrosequenced using a Pyromark Q24pyrosequencer (Qiagen) as per the manufacturer's instructions. Pyromark®Q24 software (Qiagen) was used to calculate the percent methylation foreach CpG site. The PCR products were pyrosequenced using a Pyromark Q24pyrosequencer (Qiagen) as per the manufacturer's instructions. Pyromark®Q24 software (Qiagen) was used to calculate the percent methylation foreach CpG site. The results were displayed as a pyrogram with themethylation percentage.

TABLE 1 Assays designed to evaluate CpG sites in three different geneticloci. CpG no./ Ampl. CpG sites analyzed Locus Sequence Chr. size (boldand underlined) NMUR2 Forward GTGTTGGGTAGGGAG 5 7/277 G CGCG GAA CG GGAAGAGTA (SEQ ID TGTAGGATGGTTA NO: 2) CG TAGC CG TTTTA Reverse*CTAACCTCCTAATCC CG TTGA CG GTGG TACTCCTTAAA (SEQ TGATGTTGAGGAT ID NO: 3)GGAGG Sequencing GGGTGTTTTGTAGTT (Nucleotides 85 to TG (SEQ ID NO: 4)151 of SEQ ID NO: 1/5) UBE2U Forward* GTTTTGAGATTGGGT 1 3/207 CGGTATTGTAGTG TGTGAG (SEQ ID NO: AAA CG T CG TAGA 8) TGAGGAAGTGTTT ReverseCACTTTCCCACACTT AAGTTTT AATAAACTAATA (Nucleotides 23 to 67 (SEQ ID NO:9) of SEQ ID NO: 7/11) Sequencing GATTGGGTTGTGAGT (SEQ ID NO: 10) AHRRForward TGGGGTTTTAAGGTT 5 4/233 CG AG CG TGTGAT AGGGTG (SEQ ID NO:TTTGGTGAT CG TA 14) GAGTTTTTTTGAG Reverse* AATTTCACACTTCCT GTTTT CGGGTTTT CACAATACA (SEQ ID GTGATTTTAGAAA NO: 15) GTGGT SequencingGGTGTGTTTTTTTTGT (Nucleotides 38 to AGGA (SEQ ID NO: 16) 106 of SEQ IDNO: 13/17) SA-6 Forward* AAAATATATAAATTT 2 3/124 AAACCAAT CG TCATAATTTGGAAAAGT TTCTTCCCCTACC (SEQ ID NO: 20) AAAATAAAAAA C ReverseTCTTCCCTCAAAAAA G TCCTTAT CG ACT AAATAAAACTATCC TTTCCAAATTATA (SEQ IDNO: 21) AATTTATAT Sequencing CCTAACCCCCAAATA (**:Reverse C (SEQ ID NO:22) complement of nucleotides 6 to 77 of SEQ ID NO: 19) Chr.: chromosome*: biotinylated primer no: numbers Ampl.: amplicon **: The sequenceprovided in the Table for the locus SA-6 is a reverse complement ofnucleotides 6 to 77 of SEQ ID NO: 19. Sequencing primer (SEQ ID NO: 22)is designed to sequence the double stranded amplicon produced by PCRamplification of bisulfite treated SA-6 (SEQ ID NO: 19). However, thesequence provided in the Table for the locus SA-6 is presented in thereverse complementary manner because the sequencing primer is designedto sequence the strand having a sequence complementary to the sequenceof SEQ ID NO: 19. The sequencing information can be used to sequence theamplicon corresponding to the SA-6 locus and the methylation status ofcytosines at positions 31, 40, and 68.?

HRM Analysis:

HRM analysis is a real-time PCR method that utilizes an unlabeled primerpair for amplification and includes an intercalating dye foramplification detection and melt analysis. The samples were PCRamplified using Rotor-gene SYBER Green kit (Qiagen) on a Rotor Gene 6000real-time instrument (Qiagen). The kit composed of a buffer thatconsists of SYBER Green I, HotStarTaq plus, and dNTP mix. Theamplification reaction was adjusted to 20 μl based on the total volumespecified by the manufacturer. PCR amplifications were performed byadding 1 μL of bisulfite modified DNA to a master mix that consisted of2× Rotor-Gene SYBER Green PCR master mix and 1 μM of each unlabeledforward and reverse primers. The amplifying primers utilized wereunlabeled and had the same sequence as that used for pyrosequencinganalysis. Custom designed primers were obtained from Integrated DNATechnologies (IDT), Inc. (Coralville, Iowa). PCR cycling was conductedon the GeneAmp® PCR system 9700 (Applied Biosystems, Foster City,Calif.) under the following conditions: 95° C. for 5 min; 45 cycles of95° C. for 10 s, 59° C. for 16 s, and 72° C. for 10 s. Immediatelyafterward, melt analysis was performed by increasing the temperaturefrom 65° C. to 95° C. in 0.3° C. increments and detecting fluorescencein the HRM analysis channel. Melt curve analysis was generated andmelting temperatures were determined using the Rotor-Gene 6000 seriessoftware (version 1.7).

Statistical Analysis:

Statistical analysis was performed on the methylation profiles generatedas percent methylation values from pyrosequencing or as melting pointsfrom HRM analysis. The average percent methylation value for each CpGsite identified was calculated along with the standard deviations foreach cell type. A one-way ANOVA with Tukey's test and Wetch test werecarried out to determine if there were statistical differences in thepercent methylation levels between the four types of body fluids.Methylation differences were considered statistically significant ifp-values were 0.05 or less. All the analyses were performed using SPSSstatistics software ver. 23.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1—Designing Primers for Genomic Loci Specific for Body Fluids

A set of 12 body fluid samples including blood, saliva, semen, andvaginal secretions were used for preliminary evaluation of body fluididentification using 20 different probe methylation sites.Pyrosequencing was used to perform the methylation analysis for thepreliminary evaluation at the 21 probe sites located at 12 genetic loci.Four genomic locations were found that contained differentiallymethylated regions that were tissue specific of which two were found tobe markers for sperm, one or buccal epithelial cell, and one for blood.These four body fluid markers were further screened using pyrosequencingand HRM analysis.

Pyrosequencing Data:

A total of 89 samples were used to further examine the threedifferentially methylated regions identified including 23 blood, 24saliva, 20 semen, and 22 vaginal secretion samples. The methylationprofiles were successfully analyzed using bisulfite conversion andpyrosequencing. For the loci NMUR2 and UBE2U, sperm presented lowpercent methylation (less than 20%) whereas the other body fluids hadhigh DNA methylation levels (more than 80%) (FIGS. 1 and 2). Seven CpGsites in the NMUR2 locus showed statistically significant differencesbetween the body fluids and could distinguish semen from other bodyfluids (Table 2). The UBE2U locus consisted of 3 CpG sites that showedsperm-specific methylation profiles (FIG. 2) and that showed significantdifference in methylation levels between sperm and the cells from otherbody fluids (Table 2). Both of the sperm assays provide effective toolsfor forensic identification of sperm. Three seminal samples withoutsperms produced hypermethylation patterns when tested using NMUR2 andUBE2U. The SA-6 maker consisted of 3 CpGs and found to be specific forsaliva (FIG. 3). This saliva marker showed statistical significantdifference in methylation level between the saliva samples and the otherbody fluids (Table 2). Using the saliva marker, saliva demonstratedhypermethylated patterns (more than 60%) while the other body fluidsshowed hypomethylated levels (less than 20%) (FIG. 3). The sperm freeseminal samples also showed hypomethylated patterns when tested by thesaliva marker. These results confirm that the two sperm markers and thesaliva marker and can be very effective tools for forensicidentification of sperm and saliva.

Similarly, the assay of four CpGs sites at AHHR could also be used asbiomarkers for blood identification. At the AHRR gene, blood ishypomethylated compared to other body fluid types, which have low DNAmethylation levels (FIG. 4).

TABLE 2 Mean % methylation for the pyrosequencing based assays and meanTm for the HRM analysis with the significance values based on ANOVA(p-value) and Wetch test (p-value) HRM Body Tm Marker Fluid CpG (% meanmethylation ± standard deviation) (° C.) CpG1 CpG2 CpG3 CpG4 CpG5 CpG6CpG7 All NMUR2 Semen  8.6 ± 8.1  8.4 ± 7.8  8.4 ± 8.1  9.0 ± 8.0  7.8 ±7.5  8.6 ± 7.3  8.4 ± 7.6 80.9 ± 0.1 sperm Vaginal 94.8 ± 3.8 95.0 ± 3.294.3 ± 4.0 93.5 ± 4.1 91.5 ± 3.5 92.5 ± 3.2 91.6 ± 4.0 84.6 ± 0.1Specific Secretion Saliva 96.7 ± 0.5 96.7 ± 0.6 96.1 ± 0.9 95.0 ± 1.092.4 ± 0.9 94.4 ± 1.5 93.2 ± 0.9 84.5 ± 0.2 Blood 95.8 ± 1.3 95.9 ± 1.394.4 ± 0.9 92.8 ± 1.3 87.0 ± 1.7 92.5 ± 5.1 90.9 ± 2.2 84.5 ± 0.2p-value 1.6 ×     5.5 × 10⁻⁸²    5.6 × 10⁻⁷⁹    1.0 × 10⁻⁷⁷    3.2 ×10⁻⁷⁴    3.2 × 10⁻⁸¹    6.1 × 10⁻⁷⁵    2.0 × 10⁻⁸³ 10⁻⁷⁹ p-value 1.1 ×    2.7 × 10⁻³⁶    2.1 × 10⁻³⁵    1.2 × 10⁻³⁴    1.6 × 10⁻³³    5.4 ×10⁻³⁸    5.6 × 10⁻³³    3.2 × 10⁻⁵³ 10⁻³³ UBE2U CpG1 CpG2 CpG3 All SpermSemen  5.8 ± 6.8  5.8 ± 6.6  6.0 ± 7.0 77.1 ± 0.1 Specific Vaginal 88.8± 3.1 87.5 ± 2.9 91.1 ± 2.9 78.8 ± 0.1 Secretion Saliva 92.1 ± 1.5 90.8± 1.5 93.3 ± 2.0 78.8 ± 0.1 Blood 91.1 ± 1.3 87.6 ± 2.2 91.9 ± 2.7 78.7± 0.1 p-value    9.2 × 10⁻⁸⁴    2.1 × 10⁻⁸³    4.6 × 10⁻⁸²    1.6 ×10⁻⁷⁷ p-value    1.6 × 10⁻³⁹    2.2 × 10⁻³⁹    7.8 × 10⁻⁴⁰    4.3 ×10⁻⁴⁷ SA-6 CpG1 CpG2 CpG3 All Saliva Sperm  3.5 ± 4.2  3.6 ± 3.2 1.45 ±1.1 76.4 ± 0.12 Specific Vaginal 13.3 ± 3.8   15 ± 2.8  6.2 ± 2.1 76.5 ±0.08 Secretion Saliva   75 ± 8.5 73.5 ± 7.8 76.5 ± 9.5 77.6 ± 0.05 Blood10.9 ± 2.3 14.8 ± 2.4  5.5 ± 1.5 76.4 ± 0.1  p-value    1.59 × 10⁻⁶³   5.31 × 10⁻⁶⁶    5.73 × 10⁻⁶⁸    3.38 × 10⁻⁴³ p-value    1.86 × 10⁻³²   7.55 × 10⁻³⁵    1.69 × 10⁻³⁴    1.04 × 10⁻³⁷ AHRR CpG1 CpG2 CpG3 CpG4Blood Semen 84.7 ± 11.8 88.9 ± 9.8  93.1 ± 9.8  92.7 ± 12.4 SpecificVaginal 48.2 ± 20.6 57.3 ± 20.1 62.5 ± 18.8 42.9 ± 17.1 Secretion Saliva63.0 ± 13.9 69.0 ± 13.6 73.7 ± 14.2 55.0 ± 12.9 Blood 6.5 ± 1.9 14.8 ±4.2  18.1 ± 4.0  4.7 ± 1.6 p-value    2.2 × 10⁻³¹    5.5 × 10⁻³¹    3.1× 10⁻³²    4.4 × 10⁻³⁷ p-value    1.2 × 10⁻²⁶    1.4 × 10⁻²⁸    4.3 ×10⁻²⁹    3.0 × 10⁻²⁷

HRM Analysis Data

The same set of samples consisting of 80 different body fluid types werealso used to determine whether HRM analysis would also be suited todiscriminate the methylation profiles of sperm based on CpG markers atNMUR2 and UBE2U. The melt curve representing the derivative slope offluoresce (−df/dT) over temperature for the NMUR2 marker showed adistinct pattern for sperm samples when compared to other tissues. Themelt curve showing the data for differing tissue types at the NMUR2marker is shown in FIGS. 5 and 6. At the NMUR2, the sperm samples (n=22)had a lower melting temperature averaging (80.9° C.) than those detectedfor the other tissue types including blood (84.5° C.), saliva (84.5° C.)and vaginal sections (Tm of 84.6° C.) (FIGS. 5 and 6).

In the same way, the melting curve for the UBE2U marker also showeddistinct pattern of sperm samples compared to other body fluids asillustrated in FIGS. 7 and 8. The melting temperature of sperm samplesis lower than the melting temperatures of the other three body fluids.For the UBE2U, the sperm samples (n=22) Tm averaged is 77.1° C. whilethe other tissue types had higher average melting temperature for blood(Tm=78.7° C., n=20), for saliva (Tm=78.8° C., n=21) and for vaginalsecretion (Tm=78.7° C., n=20) (FIG. 8). For the SA-6 marker, the meltingtemperatures for the saliva samples are higher than the meltingtemperatures for the cells from other body fluids (FIG. 9). FIG. 10illustrates that the average Tm is 77.6° C. for saliva samples (n=22)which is higher than the average melting temperature for blood (Tm=76.4°C., n=21), sperm (Tm=76.4° C., n=19) and vaginal secretion (Tm=76.5° C.,n=21) (Table 2). However, all four body fluids showed very similar andoverlapping melting curves and no distinct pattern can be identifiedwhen using the AHRR marker.

Example 2—Determination of the Source Body Fluids Based on the DNAMethylation Levels at Loci Specific for Body Fluids

This Example relates to identifying new tDMRs that can be used asbiomarkers for forensic body fluid discrimination. Using the discoveredtDMR, DNA methylation assays were developed based on pyrosequencing orHRM analysis.

Two novel epigenetic loci, namely NMUR2 and UBE2U, were found todifferentiate semen from other body fluids, such as blood, saliva, andvaginal secretion). The AHRR locus was able to distinguished blood fromother three tissues using the analysis of methylation signatures of eachtissue. In addition, one saliva marker (SA-6) was identified to besaliva specific in which the saliva samples were hypermethylatedcompared to the other body fluids. Thus, the intergenic region at SA-6marker located upstream from cg11536474 was able to differentiate salivawhile AHRR could distinguish blood from other three body fluids usingthe analysis of methylation signatures of each body fluid.

Neuromedin U receptor 2 (NMUR2) is a gene that encodes a protein fromG-protein coupled receptor 1 family. Such protein serves as a receptorfor neuromedin U which is a neuropeptide that is extensively distributedin the gut and central nervous system. This receptor has an importantrole in regulating food intake and body weight. UBE2U is a proteincoding gene for Ubiquitin Conjugating Enzyme E2U. This protein cancatalyze the covalent attachment of ubiquitin to other proteins. Thegenes that encode NMUR2 and UBE2U proteins appear to be very importantfor semen fluid and its components and not so for the other body fluidstested.

The AHRR gene encodes Aryl-Hydrocarbon Receptor Repressor. This proteinis involved in mediating detoxification of harmful substances such asthe toxins involved in tobacco smoking. In addition, AHRR is associatedwith regulation of cell growth and differentiation, the function thatcorrelates with the novel methylation sites identified here that canserve as a blood biomarker. The site at the saliva marker is located atintergenic location upstream from the probe site cg11536474 and is apart of the CpG island promotor region.

The results of testing three seminal fluids from vasectomized male usingpyrosequencing and HRM analysis indicate that NMUR2 and the UBE2U aresperm specific. The two sperm markers, NMUR2 and UBE2U, can effectivelydiscriminate semen markers from other body fluids using pyrosequencing(FIGS. 1 and 2). The NMUR2 assay consists of 7 CpG sites, all of whichwere hypomethylated for sperm while being hypermethylated in othertissues (FIG. 1). The UBE2U assay contains 3 CpG sites that show low DNAmethylation level for sperm compared to the other three tissues (FIG.2). The AHRR blood marker consists of 4 CpG sites that arehypomethylated for blood when compared to other body fluids (FIG. 4).

In general, all CpGs within each of the identified markers presentclearly distinguishable DNA methylation levels between the target bodyfluid and all other body fluids examined, and the differences arestatistically significant (p>0.05) (Table 2). Therefore, pyrosequencingprovides quantitative results for each individual CpG and permits thesemarkers to be utilized for forensic identification of semen, saliva, andblood samples.

However, if specific methylation values are not required, another quick,simple and inexpensive method to utilize is the HRM analysis. HRManalysis requires only a pair of unlabeled primers and the analysis canbe completed in one step. The sperm assay based on NMUR2 produces a meltcurve with lower melting temperature for sperm when compared to blood,saliva, semen without sperm, and vaginal secretions. FIGS. 5 and 6illustrate that DNA from sperm samples presents a melting temperature(Tm) of 80.9° C. with a standard deviation of 0.1° C. which is lowerthan other body fluids (84.5° C.±0.2 for blood, 84.5° C.±0.2 for saliva,and 84.5° C.±0.1 for vaginal secretion). This indicates that the meltingtemperatures for sperm samples were approximately 3.6° C. lower than themelting temperatures of the other tissue types.

For the UBE2U assay, sperm also gave a melt curve that had lower meltingthan the other body fluids. Sperm DNA demonstrated melting temperatureaverages of 77.1° C. with a standard deviation of 0.1° C., which islower than other body fluids (78.7° C.±0.1 for blood, 78.8° C.±0.1 forsaliva and 78.7° C.±0.1 for vaginal secretions) as shown in FIGS. 7 and8. This locus showed an approximately 1.6° C. difference in the meltingtemperature in the sperm samples when compared to the Tm averages of theother tissues. The fact that these two sperm markers are almost entirelyhypomethylated means that most of the cytosines were converted intothymines resulting in amplicons with low GC content and low Tms. In SA-6marker, the average Tm was 77.5° C. (+/−0.05 standard deviation) forsaliva DNA whereas other body fluids have lower average Tm (blood was76.4° C.+/−0.1, sperm was 76.4° C.+/−0.1, and vaginal secretion was76.5° C.+/−0.08). This indicates that the average Tm for saliva isdifferent by about 1° C. from the other body fluids (FIGS. 9 and 10).

The pyrosequencing data for AHRR marker showed that the DNA methylationlevel for the blood samples was 18% or below whereas the other bodyfluids had methylation levels of 48% or above (Table 2 and FIG. 4).

We claim:
 1. A method for identifying a sample as containing or notcontaining a sperm, a buccal epithelial cell, or a blood cell, themethod comprising the steps of: a) determining the DNA methylationlevels at the genetic loci corresponding to one or more of SEQ ID NOs:1, 7, 13, and 19, in: i) a genomic DNA from the sample, and ii)optionally, a control genomic DNA, through the use of one or more primerpairs selected from: i) SEQ ID NOs: 2 and 3, ii) SEQ ID NOs: 8 and 9,iii) SEQ ID NOs: 14 and 15, and iv) SEQ ID NOs: 20 and 21; b)optionally, obtaining one or more reference values corresponding to theDNA methylation levels at the genetic loci corresponding to one or moreof SEQ ID NOs: 1, 7, 13, and 19; and c) identifying the sample as: i)containing or not containing the sperm based on the DNA methylationlevel at the genetic locus comprising SEQ ID NO: 1 in the genomic DNAisolated from the sample, ii) containing or not containing the spermbased on the DNA methylation level at the genetic locus comprising SEQID NO: 7 in the genomic DNA isolated from the sample, iii) containing ornot containing the blood cell based on the DNA methylation level at thegenetic locus comprising SEQ ID NO: 13 in the genomic DNA isolated fromthe sample, and/or iv) containing or not containing the buccalepithelial cell based on the DNA methylation level at the genetic locuscomprising SEQ ID NO: 19 in the genomic DNA isolated from the sample. 2.The method of claim 1, wherein the sample comprises vaginal epithelialcells, sperms, semen cells other than sperms, blood cells, buccalepithelial cells, or a combination thereof.
 3. The method of claim 1,wherein the DNA methylation levels at the specific loci in the genomicDNA obtained from the sample are determined by high-resolution melt(HRM) analysis, wherein the HRM analysis comprises the steps of: a)isolating the genomic DNA from the sample and optionally, the controlsample; b) treating the isolated genomic DNA with bisulfite; c)polymerase chain reaction (PCR) amplifying the genetic locicorresponding to one or more of SEQ ID NOs: 1, 7, and 19 to produce thecorresponding one or more amplicons, d) determining the meltingtemperatures of the one or more amplicons; and e) identifying the sampleas: i) containing or not containing the sperm based on the meltingtemperature of the amplicon corresponding to the genetic locuscomprising SEQ ID NO: 1 in the genomic DNA isolated from the sample, ii)containing or not containing the sperm based on the melting temperatureof the amplicon corresponding to the genetic locus comprising SEQ ID NO:7 in the genomic DNA isolated from the sample, and iii) containing ornot containing the buccal epithelial cell based on the meltingtemperature of the amplicon corresponding to the genetic locuscomprising SEQ ID NO: 19 in the genomic DNA isolated from the sample. 4.The method of claim 3, wherein PCR amplifying is performed with two ormore primers in one reaction.
 5. The method of claim 1, wherein the DNAmethylation levels at the specific loci in the genomic DNA obtained fromthe sample are determined by a sequencing analysis, wherein thesequencing analysis comprises the steps of: a) isolating the genomic DNAfrom the sample and optionally, the control sample; b) treating theisolated genomic DNA with bisulfite; c) PCR amplifying the genetic locigenetic loci corresponding to one or more of SEQ ID NOs: 1, 7, 13, and19 to produce the corresponding one or more amplicons, d) determiningthe sequences of the one or more amplicons; and e) identifying thesample as: i) containing or not containing the sperm based on thesequence of the amplicon corresponding to the bisulfite treated geneticlocus comprising SEQ ID NO: 1 in the genomic DNA isolated from thesample, ii) containing or not containing the sperm based on the sequenceof the amplicon corresponding to the bisulfite treated genetic locuscomprising SEQ ID NO: 7 in the genomic DNA isolated from the sample,iii) containing or not containing the blood cell based on the sequenceof the amplicon corresponding to the bisulfite treated genetic locuscomprising SEQ ID NO: 13 in the genomic DNA isolated from the sample,and/or iv) containing or not containing the buccal epithelial cell basedon the sequence of the amplicon corresponding to the bisulfite treatedgenetic locus comprising SEQ ID NO: 19 in the genomic DNA isolated fromthe sample.
 6. The method of claim 5, wherein the sequencing analysis ofthe one or more amplicons is performed by a sequencing primer, whereinthe sequencing primer is designed based on the sequence of SEQ ID NO: 1,7, 13, or 19 and/or the sequences of the regions flanking the geneticlocus corresponding to the sequence of SEQ ID NO: 1, 7, 13, or
 19. 7.The method of claim 5, wherein PCR amplifying is performed with two ormore primer pairs in one reaction.
 8. The method of claim 5, wherein: i)the sequencing analysis of the amplicon corresponding to the bisulfitetreated genetic locus comprising SEQ ID NO: 1 is performed using asequencing primer comprising SEQ ID NO: 4, ii) the sequencing analysisof the amplicon corresponding to the bisulfite treated genetic locuscomprising SEQ ID NO: 7 is performed using a sequencing primercomprising SEQ ID NO: 10, iii) the sequencing analysis of the ampliconcorresponding to the bisulfite treated genetic locus comprising SEQ IDNO: 13 is performed using a sequencing primer comprising SEQ ID NO: 16,and/or iv) the sequencing analysis of the amplicon corresponding to thebisulfite treated genetic locus comprising SEQ ID NO: 19 is performedusing a sequencing primer comprising SEQ ID NO:
 22. 9. A method fordetermining the DNA methylation levels at genetic loci corresponding toone or more of SEQ ID NOs: 1, 7, 13, and 19, in a genomic DNA isolatedfrom a human cell, the method comprising determining the DNA methylationlevels at genetic loci by HRM analysis, or determining the DNAmethylation levels at genetic loci by a sequencing analysis, using oneor more primer pairs selected from: i) SEQ ID NOs: 2 and 3, ii) SEQ IDNOs: 8 and 9, iii) SEQ ID NOs: 14 and 15, and iv) SEQ ID NOs: 20 and 21.10. The method of claim 9, wherein the DNA methylation levels at thespecific loci in the genomic DNA obtained from the human cell aredetermined by HRM analysis, wherein the HRM analysis comprises the stepsof: a) isolating the genomic DNA from the cell; b) treating the isolatedgenomic DNA with bisulfite; c) PCR amplifying the genetic locicorresponding to one or more of SEQ ID NOs: 1, 7, and 19 to produce thecorresponding one or more amplicons; d) determining the meltingtemperatures of the one or more amplicons; and e) determining the DNAmethylation levels at the genetic loci based on the melting temperaturesof the one or more amplicons.
 11. The method of claim 10, wherein PCRamplifying is performed with two or more primers in one reaction. 12.The method of claim 9, wherein the DNA methylation levels at thespecific loci in the genomic DNA obtained from the sample are determinedby a sequencing analysis, wherein the sequencing analysis comprises thesteps of: a) isolating the genomic DNA from the cell; b) treating theisolated genomic DNA with bisulfite; c) PCR amplifying the genetic locicorresponding to one or more of SEQ ID NOs: 1, 7, 13, and 19 to producethe corresponding one or more amplicons; and d) determining thesequences of the one or more amplicons.
 13. The method of claim 12,wherein the sequencing analysis is performed by pyrosequencing.
 14. Themethod of claim 13, wherein the sequencing analysis of the one or moreamplicons is performed by one or more sequencing primers, wherein thesequencing primers are designed based on the sequences of SEQ ID NO: 1,7, 13, or 19 and/or the sequences of the regions flanking the geneticloci corresponding to the sequences of SEQ ID NO: 1, 7, 13, or
 19. 15.The method of claim 14, wherein: i) the sequencing analysis of theamplicon corresponding to the bisulfite treated genetic locus comprisingSEQ ID NO: 1 is performed using a sequencing primer comprising SEQ IDNO: 4, ii) the sequencing analysis of the amplicon corresponding to thebisulfite treated genetic locus comprising SEQ ID NO: 7 is performedusing a sequencing primer comprising SEQ ID NO: 10, iii) the sequencinganalysis of the amplicon corresponding to the bisulfite treated geneticlocus comprising SEQ ID NO: 13 is performed using a sequencing primercomprising SEQ ID NO: 16, and/or iv) the sequencing analysis of theamplicon corresponding to the bisulfite treated genetic locus comprisingSEQ ID NO: 19 is performed using a sequencing primer comprising SEQ IDNO:
 22. 16. The method of claim 3, wherein the PCR amplifying isperformed using primer pairs: i) SEQ ID NOs: 2 and 3, ii) SEQ ID NOs: 8and 9, and iii) SEQ ID NOs: 20 and
 21. 17. The method of claim 5,wherein the PCR amplifying is performed using primer pairs: i) SEQ IDNOs: 2 and 3, ii) SEQ ID NOs: 8 and 9, iii) SEQ ID NOs: 14 and 15, andiv) SEQ ID NOs: 20 and
 21. 18. The method of claim 10, wherein the PCRamplifying is performed using primer pairs: i) SEQ ID NOs: 2 and 3, ii)SEQ ID NOs: 8 and 9, and iii) SEQ ID NOs: 20 and
 21. 19. The method ofclaim 12, wherein the PCR amplifying is performed using primer pairs: i)SEQ ID NOs: 2 and 3, ii) SEQ ID NOs: 8 and 9, iii) SEQ ID NOs: 14 and15, and iv) SEQ ID NOs: 20 and
 21. 20. The method of claim 3, whereineach primer pair is labelled with a fluorophore.